Chromatin immunoprecipitation (ChIP) is a powerful tool for investigating the mechanisms through which nuclear proteins influence gene regulation. ChIP can be used to determine whether or not a particular protein is present in the in the chromatin of a living cell, to localize the region of interaction of a protein within the genomic DNA, and to isolate chromatin fragments that contain a particular target. However, since the basis of ChIP is the recognition of particular proteins or functional groups, it is limited in its ability to interrogate the full complement of proteins in a specific genomic region. In order to overcome this limitation, we propose to develop new approaches to analysis of chromatin that uses hybridization probes to target specific DNA sequences rather than antibody-based recognition. Our particular interest is in genomic DNA that may form alternate structures during nuclear processes, specifically in this application in gene promoter regions that contain G-rich sequences known to form G-quadruplex structures in vitro. These regions are the focus of much speculation and growing investigation in the cancer research community. The specific aims for this project period are to (1) demonstrate feasibility and selectivity and optimize conditions for in vivo capture of target chromatin DNA and associated proteins using chromatin antisense rehybridization (CAR) and chromatin sense rehybridization (CSR) with the model system of the insulin- linked polymorphic region (ILPR), and (2) evaluate the effectiveness of CAR and CSR to probe the proteins associated with the human c-myc oncogene promoter region in both cancer and normal cells. The proposed CAR/CSR techniques should be adaptable to isolation of target DNA and associated proteins in any genomic region of interest, and will be an important addition to the chromatin analysis toolbox. They will complement ChIP techniques by targeting specific DNA sequences rather than specific antibody targets such as proteins, thereby enabling discovery of new protein interactions that participate in gene regulation. There is exciting potential for discovery in epigenetic regulation as well. We propose to develop new approaches to analysis of chromatin, the genetic material in the nucleus of the living cell that uses hybridization probes to target specific DNA sequences rather than antibody-based recognition of immunochemical targets. Identification of the full complement of proteins associated with a particular DNA target of interest such as a gene promoter region in chromatin will lead to a better understanding of gene regulation and to the discovery of new biomarkers and drug targets.